Flavivirus Vaccine

ABSTRACT

Replicon virus-like particles can be used as flavivirus vaccines.

BACKGROUND OF THE INVENTION

The instant invention relates to the Flaviviridae family of viruses. Theflavivirus genus includes about 70 members, 40 of which are associatedwith human illness. The majority of flaviviruses are arboviruses,transmitted to their avian and mammalian hosts, including man, bymosquitoes or ticks. Dengue fever virus (types 1-4), yellow fever virus,Japanese encephalitis virus, tick-borne encephalitis virus and West Nilevirus are causative agents of significant morbidity and mortality inhuman populations. Several flaviviruses, including louping ill thatcauses neurological disease in sheep, West Nile that causes encephalitisin horse, and Japanese encephalitis that also causes encephalitis inhorses as well as stillbirth in domestic pigs, are important veterinarypathogens.

Flavivirus genomes consist of a single linear, single-stranded+senseRNA. The +strand is capable of infecting appropriate host cells. Thetotal genome can range from 10 to 11 kbs. There is no 3′polyadenylation. The 5′ end has a methylated cap.

Flavivirus genomes do not contain internal ribosomal entry sites (IRES)that provide a site of translation initiation for host ribosomes.Instead flavivirus employs ribosomal scanning to commence proteinsynthesis.

Flavivirus virions are spheres, 40-65 nm in diameter. Under the lipidenvelope is an icosahedral capsid coat approximately 25-30 nm indiameter.

Generally, flaviviruses are transmitted by arthropods, for example,mosquitoes and ticks. Flaviviruses reproduce in their vector organismand are passed from one host to the next.

The yellow fever virus is capable of causing large epidemics. The yellowfever virus is transmitted in monkey and human hosts and in mosquitoes.In the first cycle, the virus is transmitted by Aedes africanus andother Aedes mosquitoes (in Africa) or by Hemogogus mosquitoes (in theAmericas); monkeys serve as the reservoir, and generally, humansinfected are those who enter deep forests and jungles. In the secondcycle, the domestic mosquito, Aedes aegypti, which lives in closerelationship with humans, may transmit the virus directly to humans, thesole host in the cycle.

The tick-borne encephalitis virus is transmitted by ticks of the genusIxodes in temperate regions of Russia and Europe. The virus can onlyaffect humans in areas where the ticks exist.

Flaviviruses cause other encephalitic diseases, such as, Murray Valleyencephalitis, Rocio and Powassan encephalitis, and as more recentlyobserved in North America, West Nile fever.

Dengue fever is an acute infectious disease characterized by biphasicfever, headache, pain in various parts of the body, prostration, rash,lymphadenopathy and leucopenia (Holstead, S B, 1980, Immunologicalparameters of togavirus disease syndromes, p. 107-173, in R WSchlesinger (ed.) The Togaviruses, Academic Press, Inc., NY; Sabin, A B,1959, Dengue, p. 361-373, in T Rivers and F Horsfall (eds.), Viral andRickettsial Infections of Man, JB Lippincott Co., Philadelphia).

Dengue is mosquito-borne and caused by four serologically relatedviruses known as dengue virus type 1 to type 4 (dengue-1 to dengue-4).Infection with one dengue serotype provides lifelong immunity to thatsubtype, but no cross-protective immunity to the other serotypes. Thus,persons living in an area of endemic dengue can be infected with three,and possibly four, dengue serotypes during their lifetime. Illnessranges from unapparent infection to dengue fever or, in severe cases,potentially fatal dengue hemorrhagic fever/dengue shock syndrome(DHF/DSS).

Dengue hemorrhagic fever (DHF) is a severe febrile disease characterizedby abnormalities of hemostasis and increased vascular permeability,which in some instances results in a hypovolemic shock syndrome, dengueshock syndrome (DSS) (World Health Organization: 1975. Technical Guidesfor Diagnosis, Treatment, Surveillance, Prevention and Control of DengueHemorrhagic Fever. World Health Organization. Geneva). The mechanism ofDHF/DSS may vary in different cases. The major factors contributing toDHF/DSS may include viral virulence, patient health status and secondaryinfection of different serotype dengue virus.

Considering the urgent need for flavivirus vaccines, a novel alternativeapproach is needed. Theoretically, live attenuated vaccines elicit themost effective, long-term, virus-specific immunity, and inactivatedvirus vaccines, including recombinant subunit vaccines, provide thehighest level of safety. The ideal vaccine would be the one that canproduce the efficacy of a live vaccine and the safety of subunitvaccine. That goal was met in the development of the pseudoinfectiousvirus-like particle (PVLP) flavivirus vaccines described herein.

SUMMARY OF THE INVENTION

The inventions relates to materials and methods for making a flavivirusvaccine. That vaccine comprises a virus-like particle (VLP) that caninfect a host cell, can replicate in that host cell to produce viralproteins that are recognized by the host immune system, but which cannotbe packaged into infectious viral particles. The virus-like particles ofinterest contain a replicon that has all of the necessary genes toensure replicon RNA production and production of the flaviviralproteins. Thus, a preferred replicon is one that contains all of thenon-structural genes, the preM and E genes. However, the capsid gene ismanipulated to ensure that capsid protein either is not made, or what ismade cannot be used to make an infectious viral particle, and to ensurehigh level expression of the preM and E proteins. The virus-likeparticles of interest can infect a host cell, then multiple copies ofthe replicon are made in the cell and the replicon is expressed toproduct preM and E proteins. The preM and E proteins can be releasedfrom the cell or expressed at the surface of the cell, and thus thevaccine of interest elicits both a host humoral and cellular response.

Additional features and advantages of the present invention aredescribed in, and will be apparent from, the following DetailedDescription of the Invention and the figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts the architecture of a flavivirus.

FIG. 2 depicts the flavivirus genome, and translation and processing ofthe Flavivirus polyprotein. At the top is the viral genome with thestructural (S) and nonstructural (NS) protein coding regions, the 5′cap, putative 3′ secondary structure, and the 5′ and 3′ non-translatedregions (NTR) indicated. Boxes below the genome indicate precursors andmature proteins generated by the proteolytic processing cascade. Maturestructural proteins are indicated by shaded boxes, and the nonstructuralproteins and structural protein precursors by open boxes. Contiguousstretches of uncharged amino acids are shown by black bars. Asterisksdenote proteins with N-linked glycans but do not necessarily indicatethe position or number of sites used. Cleavage sites for proteases areindicated. ORF is open reading frame.

FIG. 3 diagrams the genome of dengue virus and derived replicons. Thereplicon DEN2/ΔprM-E was made from deletion of pre-membrane (prM)protein and envelope (E) protein from nucleotide 452 to nucleotide 2340.The replicon DEN2/ΔC-prM-E was made from deletion of nucleotide 157 tonucleotide 2340. The replicon DEN2/ΔC was made from deletion ofnucleotide 160 to nucleotide 320. UCR is non-coding region.

FIG. 4 depicts a protocol for making full length cDNA flavivirus clonesusing a shuttle vector system. TRP representsphosphoribosyl-anthranilate isomerase, a selectable marker gene ofyeast; 2 μm represents the yeast origin of replication from thatplasmid; amp is the ampicillin resistance gene; and ori represents abacterial origin of replication. RT-PCR is reverse transcriptasepolymerase chain reaction.

FIG. 5 depicts a construct of an NS3-deleted flavivirus subgenomeexpression plasmid. CMV is the cytomegalovirus promoter. UTR is anuntranslated region. HDVr is described in the text. Neo is the neomycinresistance gene. pA is a polyadenylation site.

FIG. 6 depicts a scheme for production of construct. The pTet-offplasmid was used to generate a first stable cell line, BHK/Tet-off,autoregulatedly expressing the tetracycline transactivator (tTA). TRE,tetracycline-responsive element. P_(minCMV), the minimal CMV promoter.tTA, tetracycline transactivator. IRES, internal ribosome entry site ofencephalomyocarditis virus. Hyro, hygromycin B phosphotransferase gene.Neo, neomycin phosphotransferase gene. Intron is a synthetic sequencefound in certain vectors of Clontech. Cpr is a polypeptide fragment thatcontains capsid protein and a segment of the pre portion of thepremembrane protein.

DETAILED DESCRIPTION OF THE INVENTION

The flavivirus virion is composed of 6% RNA, 66% protein, 9%carbohydrate and 17% lipid (Russell P K, Brandt W E, Dalrymple J M,Chemical and Antigenic Structure of Flaviviruses, in Schlesinger R W,eds., The Togaviruses: Biology, Structure, Replication. New York:Academic; 1980: 503-529; and Trent D W, Naeve C W, Biochemistry andReplication, in Monath T, ed. St. Louis Encephalitis. Washington, D.C.:American Public Health Association; 1980 p. 159-199). An electron-densenucleocapsid is composed of C (capsid) protein and genomic RNA. Theenvelope protein, E, and membrane (M) protein are embedded in the lipidbilayer by C-terminal hydrophobic anchors. However, immature particlesfound within intracellular vesicles contain exclusively unprocessed prMand are less infectious than released virions (Morens D M:Antibody-dependent enhancement of infection and the pathogenesis ofviral disease. Clin Infect Dis 1994, 19:500-512).

The genome of flaviviruses is uniform, and is a single-stranded,positive-sense RNA molecule of about 10-11 kb, containing a single ORFconstituting roughly 95% of the genome (Chambers T J, Hahn C S, GallerR, Rice C M: Flavivirus genome organization, expression, andreplication, Ann Rev Microbiol 1990, 44:649-688). Genome-length RNAsappear to be the only virus-specific messenger RNA (mRNA) molecules indengue-infected cells. On infection, the viral RNA is translated into apolyprotein of about 3400 amino acids that is processed into 10 geneproducts: the three structural proteins C, prM and E, and sevennonstructural (NS) proteins, 1, 2A, 2B, 3, 4A, 4B and 5 (BhamarapravatiN, Yokan S: Live attenuated tetravalent vaccine, in Gubler D J, Kuno G(eds.): Dengue and Dengue Hemorrhagic Fever. Wallingford, CABInternational, 1997, pp 367-377; and Falgout, B and Markoff, L, 1995,The family flaviviridae and its diseases, p. 47-66. in: J S Porterfield(ed.), Exotic Viral Infections. Chapman and Hall Medical, London, UnitedKingdom).

As for all positive-stranded RNA virus, flavivirus genomic RNA isinfectious. A focus of the instant invention is to manipulate the genometo produce replicons of a flavivirus that are defective in producing aninfection, but continue to express those epitopes and determinants thatcan be recognized by the host, such as expressed by M and E genes.

For example, replicons can be constructed where portions orsubstantially all of the structural genes are deleted. Thus, some or allof C, prM and/or E can be removed. In the case of C, for example, nearlyall of C, as few as only 20 remaining amino acids, can be removedwithout impacting replication and expression.

On the other hand, the instant invention relates to defective viralgenomes that contain a majority of the structural proteins, or at theleast, the majority of the polypeptide structures that are determinantsfor antibodies generated by the host. Thus, it is preferable that mostof prM and E coding sequences are present as the proteins expressedthereby are the primary immunogenic sites in the wild-type virusinfection. The C protein is less of a target for the host immune systemso C would be the more favored target for manipulation to render thevirus incapable of replication.

Thus, the C coding sequence can be deleted in part or in whole. The Ccoding sequence can be altered in other ways, such as with one or morepoint mutations, inversions, deletions and the like to ensure that thecapsid protein is not expressed or the capsid protein that is expressedcannot be used to make a functional capsid for an infectious virus,without negatively impacting the expression of the preM and E codingsequences. Thus, a capsid can be made that contains additional expressedamino acids that prevent the protein from either folding properly orfrom being able to make the proper shell for the replicon.

Various dengue replicons were constructed as shown in FIG. 3, which havevarying deletions and modifications of the structural genes. Whenintroduced into host cells by electroporation, replicon replicationoccurred and expression occurred. The particle of interest is one whichwhen made as taught in the instant invention yields an infectiousparticle but containing a defective replicon that cannot be packagedinto infectious particles in host cells. Thus, a particle of interestinfects once, the replicon is replicated and expressed, but the repliconis not packaged into particles.

A favored type of vector would be of the nature and structure as the onedepicted in the lowermost diagram of FIG. 3, wherein only C is renderednon-expressible. Because of the similarity of genomic structures amongthe flaviviruses, that same approach is taken with other serotypes,strains and species of flavivirus, such as Japan encephalitis, West Nilevirus and yellow fever.

Flavivirus infectious clones can be unstable in Escherichia coli. Thathurdle can be overcome by using eukaryotic host cells such asSaccharomyces cerevisiae. Shuttle vectors using dengue virus infectiousfull-length clones have been made (Polo, S, Ketner, G, Levis, R andFalgout, B, 1997, Infectious RNA transcriptions from full-length denguevirus type 2 cDNA clones made in yeast. J Virol 71:5366-5374; Pang, Xand Markoff, L 1998, A full-length “infectious” cDNA clone of a dengueserotype 2 vaccine virus. Poster, Fifth International Symposium onPositive Strand RNA viruses. p. 1-73; and Pur, B, Polo, S, Hayes, C,Falgout, B, 2000, Construction of full length infectious clone fordengue-1 virus western pacific 74 strain. Virus Genes, 2000: 20(1):57-63). The shuttle vector contains a bacterial replication origin andselectable marker, and a yeast replication origin and a yeast selectablemarker.

Thus, to assist in the construction and scale up production of a PVLP ofthe instant invention, a scheme is constructed wherein cloning of theflavivirus 5′ and 3′ cDNA fragments lacking a central portion occurs ina polylinker of the shuttle vector, and then in yeast, a full-lengthcDNA clone is assembled by homologous recombination between the centralcDNA fragment of the Flavivirus genome and the cloned 5′ end and 3′ end,as depicted in FIG. 4.

The replicon with some portion of the C gene deleted can express allmajor viral antigens and is most immunogenic of the replicons made andtested. The instant invention is related to developing a flavivirusreplicon which when expressed, contains the most immunogenicity. Thus, areplicon of interest is one that contains most or all of the known viralantigens, one which has one or more defects that prevent viralinfectivity yet enable continued RNA replication and expression withinthe host cells because the replicon cannot be packaged into VLP in hostcells.

The replicon of interest also is one that does not necessarily contain atransgene as the purpose of the replicon is to produce preM and Eprotein. Thus, the replicon of interest is not one targeted to carryingforeign genes, that is, the replicon is not a cloning vector. Instead, areplicon of interest is targeted to contain as much of the flavivirusgenome without being replication competent, and not containing any genesof another virus or another species of flavivirus. However, the repliconcan be constructed to contain and express a molecule that can be used toenhance host recognition and reaction to the preM and E proteins. Thus,the replicon can be configured to contain, for example, an adjuvant orany other molecule that enhances immunogenicity.

Moreover, changes can be made to the preM and E coding sequences toensure or to enhance immunogenicity of same. Thus, point mutations andthe like can be made in the preM and E coding sequences so that whenexpressed in a host, the expressed proteins result in animmunoprotecting response.

With that in mind, there are several methods available to the artisan topackage the replicon of interest into a virus-like particle. Generally,the strategies employ the use of additional vectors or packaging cellsthat provide the necessary components in trans that can complement thedefective replicon to enable packaging into a particle.

Thus, another goal of the instant invention is to develop a packagingsystem for a flavivirus replicon. For example, a Sindbis repliconcapable of expressing dengue structural proteins prM, E and C wasconstructed. When the Sindbis replicon transfected cells twenty-fourhours after defective dengue replicon transfection, dengue replicon RNAwas encapsidated in “virus-like” particles (VLPs) and released inculture medium.

Thus, replicon RNA can be packaged into a VLP when structural proteinsare supplied in trans using an appropriate packaging cell containing theappropriate complementing expression products.

Since the replicon containing VLP is infectious, but cannot produceinfectious virions from the infection, because, for example, propercapsid proteins are not expressed in the infected host cell, the VLP isnamed as a “pseudoinfectious virus-like particle (PVLP) or anon-replicating particle. Thus, a non-replicating particle is one thatinfects a host cell, but that host cell does not yield infectious virusparticles resulting from that infection. For the purposes of the instantinvention and the teachings herein, PVLP and VLP are synonymous.

The unique character of the PVLP makes the PVLP a source for effectiveand safe flavivirus vaccines. A highly immunogenic viral strain of PVLPshould mimic the process of natural infection of a flavivirus andproduce long-lasting immunity. Thus, a preferred PVLP of interest is onethat contains the genetic material to express all of the epitopesexpressed by the wild-type prM and E proteins, and perhaps, C as well.If manipulations are to be made to the structural proteins to ensurereplication incompetence, changes should be made to the C protein, and alesser amount of change is preferred.

The first embodiment of a packaging system uses two consecutiveinfections: first with a Flavivirus PVLP, and 24 hours later, with aSindbis PVLP that provides Flavivirus structural proteins in trans, asdescribed in further detail below. The development of alphaviruspackaging cell lines has been described (Polo, J M, Belli, B, Driver, D,Frolov, I, Sherrill, S, Hariharan, M J, Townsend, K, Perri, S, Ment, SJ, Jolly, D J, Chang, S W, Schlesinger, S and Dubensky, Jr, T, 1999.Stable alphavirus packaging cell lines for Sindbis virus-derived vectorsand Semliki forest virus-derived vectors. Proc Natl Acad Sci USA Vol96:4598-4603). The Sindbis virus packaging cell line of interest employstwo expression cassettes each containing a part of the structural genes.Some modifications can be made to facilitate the construction ofcassettes and to enhance packaging efficiency.

Flavivirus PVLP infect up to 100% of susceptible cells followingappropriate titration. Vero cells in a six-well dish are infected withPVLP at serial dilution. About 24 hours post-infection, packagingSindbis PVLP are added into each six-well dish, and agitated in a rockerplatform for 2 hours at 37° C., and then the unattached Sindbis PVLPwere washed out with culture medium. Cell culture fluid is collected at48 hours after the second infection and treated with Sindbis-specificpolyclonal antibodies to remove possible contaminants ofreplicative-competent Sindbis particles.

To prepare partially purified PVLP, culture fluid is clarified bycentrifugation at 16,000×g in a microcentrifuge for 15 min at 40° C.,and the particles are pelleted from the supernatant fluid byultracentrifugation at 40,000 rpm for 2 hours at 40° C. in the AH650rotor of a Sorvall OTD55B centrifuge. The pellets are resuspended in 50μl of PBS and left to dissolve overnight at 40° C. To determine thetiter of the dengue PVLPs, BHK-21 cells on eight-well chamber slides areinfected with 50 μl of serial 10-fold dilutions of cell culture fluid orof resuspended pelleted material for 2 hours at 37° C. The fluid then isreplaced with 1 ml of Dulbecco's minimal essential medium supplementedwith 2% fetal bovine serum. Cells are incubated for 24 hours at 37° C.in a CO₂ incubator and subjected to immunofluorescence (IF) analysiswith a 1:100 dilution of HMAF as described below.

The second embodiment of a packaging cell line is based on a mammaliangene expression system. The eukaryotic expression plasmid pCI-neo(Promega) is used for the expression of structural protein C-pr (prrepresents the pr segment of prM), due to its high expression rate(Almond, B D and Schenborn, E T, A comparison of pCI-neo vector andpcDNA4/HisMax vector. Promega Publication). The pCI-neo mammalianexpression vector contains a CMV IE enhancer/promoter, an optimizedchimeric intron and the simian virus 40 (SV40) late polyadenylationsignal. Those three elements combine to yield strong, constitutiveexpression of the cloned gene in mammalian cells. For example, as todengue virus type 2 strain, the gene fragment encoding aa 1 to 205 (nt93-640) of the dengue-2 NGC strain virus structural protein C and pr isPCR amplified using the sense primer and the antisense primer, withdengue-2 cDNA as the template. Unique restriction sites, XhoI and XbaI,are created in the primers. The PCR product as well as pCI vector aredigested with XhoI and XbaI, and then purified by Qiagen column. Thepurified XhoI/XbaI-digested PCR product and pCI fragments are ligated toform the pCI-C plasmid. The plasmid DNA from the selected clone ispurified by Qiagen column. The sequence of protein C gene is confirmedby DNA sequencing. After BHK-21 cells are transfected with pCI-C byelectroporation, individual clones of G418-resistant cells are selectedby limited dilution in a 96-well dish. Dengue-2 replicon with theprotein gene C gene deleted can be used to transfect the cloned cells,and culture fluid collected to determine the packaging ability of eachclone as described above. Favored packaging cell lines are those thatproduce the highest levels of virus-like particles free fromreplication-competent virus.

The third embodiment of a packaging cell line is based on thetetracycline induced gene expression system. The system is commerciallyavailable from Clontech in Tet-On and Tet-Off formulations, allowinginduction of gene expression either by addition or by the removal oftetracycline, see Clontech manuals for a depiction of the constructs.The Tet-Off system was chosen to avoid the presence of antibiotic inPVLP preparations. To generate a packaging cell line that allowstetracycline-inducible expression of the flavivirus structural genecassette (Cpr), a BHK cell line, BHK-Tet-Off, stably expressing thetetracycline transactivator was established. Thus, BHK21 cells weretransfected with a pTet-off plasmid DNA. Two days followingtransfection, the antibiotic G418 (Sigma) was added at a concentrationof 200 μg/ml for selection of cell clones. Cell clones were isolated andcultured successfully from this transfection. These clones were thenanalyzed for induction of expression by transfection with the plasmid,pTRE2luciferase (Clontech), in the presence (0.5 μg/ml) or absence ofdoxycycline (an antibiotic of the same spectrum as tetracycline but withhigher specific activity and a longer half-life). The BHK-Tet-Off cellclone displaying the highest fold induction of luciferase expressioncompared to uninduced cells were used to establish a stable BHK cellline expressing the flavivirus structural gene cassette Cpr. The cellswere transfected with pTRECpr plasmid DNA constructed by cloning the PCRfragment of a flavivirus Cpr gene and pTK-Hyg plasmid. Transfected cellswere subjected to selection with 10 μg/ml of hygromycin in medium thatalso contained 200 μg/ml of G418 and 0.5 μg of doxycycline/ml. To selectthe most efficient packaging cell line, a number of cell clonesconferring resistance to G418 and puromycin were electroporated with aflavivirus replicon RNA and cultured without doxycycline to determinewhether they were able to produce PVLPs. The titers of PVLPs (ininfectious units [IU] per milliliter) present in harvested culturefluids (CFs) were determined by infection of Vero cells followed byimmunofluorescence analysis with mouse hyperimmune acidic fluid. Themost efficient cell clone was used for PVLP production.

In another embodiment, as a variant of the tetracycline induciblesystem, two sets of bicistronic expression vectors, pTet-off and pCpr(FIG. 6), were constructed for increasing the stability of the packagingcell line and reducing the required numbers of screening cells. Theautoregulated expression plasmid, pTet-off, was constructed with atetracycline-regulatable promoter, P_(hcMV*-1) (from plasmid pTRE2 ofClontech), and followed by bicistronic expression cassette of thetetracycline-responsive transcriptional activator (tTA) and hygromycin Bphosphotransferase (Hyg^(r)). The autoregulatory tTA expression vectorallows higher levels of tTA expression when induced, while minimizes thetoxic effect of tTA by keeping low level expression of tTA withdoxycycline.

Plasmid pCpr was constructed with the promoter P_(hcMV*-1) followed by abicistronic expression cassette of C-pr and Neomycin phosphotransferase(Ned).

BHK/Tet-off cells were developed by transfection of BHK-21 cells withplasmid pTet-off, and subjected to selection with 0.4 mg/ml ofhygromycin B and 0.5 μg/ml of doxycycline. Ten clones were analyzed forinducible expression by transfection with plasmid pTRE2EGFP (Clontech),in the presence (0.5 μg/ml) or absence of doxycycline. All clones showedsome degree of inducibility for EGFP expression. The cell clone withhighest inducible production of EGFP was used to establish a stable BHKcell line to express dengue protein, C-pr. The cells were transfectedwith plasmid pCpr and subjected to selection with 0.2 mg/ml G418 and 0.3mg/ml of hygromycin B, as well as 0.5 μg/ml of doxycycline. To selectthe most efficient packaging cell line, fifteen cell clones wereelectroporated with replicon DEN2/AC (a clone with deletion in the Ccoding sequence from nucleotide 160 through 320) RNA and cultured inmedium without doxycycline.

The titers of replicon VLP (in infectious units (IU) per milliliter)present in harvested culture fluids (CFs) were determined by infectionof LLC-MK2 cells followed by indirect immunofluorescence analysis (IF)with dengue type 3 hyperimmune mouse ascitic fluid (HMAF). Ten of 15clones produced replicon VLPs. The most efficient cell clone,BHK/C-pr/8, producing 4.6×10⁶ IU of replicon VLPs.

The fourth embodiment of a packaging cell line is based ontranscomplementation of defective flavivirus genomic RNAs with a largelethal deletion in the C-terminal region of a nonstructural gene, suchas NS3.

The plasmid was prepared by replacing the SP6 promoter in thefull-length clone described above with the cytomegalovirus (CMV)-derivedimmediate-early enhancer/promoter region. The fragment containing theCMV sequence followed by the 5′ end of the flavivirus sequence wasproduced in a fusion PCR. The hepatitis delta virus antigenomic ribozyme(HDVr) sequence was inserted immediately downstream of the lastnucleotide of the flavivirus sequence. The encephalomyelocarditis virusinternal ribosome entry site (IRES) sequence and neomycin resistancegene (ORF) were added for selection. BHK21 cells were transfected withthe plasmid DNA. Two days following transfection, the antibiotic G418(Sigma) was added at a concentration of 200 μg/ml for selection of cellclones. Cell clones were isolated and cultured successfully from thistransfection. To select the most efficient packaging cell line, a numberof cell clones conferring resistance to G418 and puromycin wereelectroporated with flavivirus replicon RNA to determine whether theywere able to produce PVLPs. The titers of PVLPs (in infectious units[IU] per milliliter) present in harvested culture fluids (CFs) weredetermined by infection of Vero cells followed by immunofluorescenceanalysis with mouse hyperimmune acidic fluid. The most efficient cellclone was used for PVLP production.

To further facilitate the formation of virus-like particles (VLP), aminoacid insertions and substitutions (for example, the VPQAQA mutation) atthe COOH terminus of the prM signal sequence can be used (see FIG. 3,uppermost diagram, the VPQAQA mutation could be inserted at residues109-114 at the juncture of the C and prM domains). The VPQAQA sequenceis one that is known to enhance signal peptidase cleavage. The insertionfacilitates efficient signal peptidase cleavage of the prM protein fromits dependence on cleavage in the cytoplasm by the viral NS2B-3protease. The result is enhanced packaging and packaging efficiency.

Flavivirus RNA replicons were encapsidated into a PVLP by a procedureinvolving two consecutive electroporations of cells, first, for example,with flavivirus replicon RNA and about twenty-four hours later with arecombinant Sindbis virus replicon RNA expressing transcomplementingflavivirus structural proteins. Once the flavivirus PVLPs are obtained,the first cell electroporation by the flavivirus replicon RNA can bereplaced by an infection of the PVLP. The use of Sindbis to deliverflavivirus genes in trans has been used in the production ofpicornovirus (e.g. poliovirus) replicon PVLPs with the necessarystructural proteins in trans using a vaccinia vector (Porter, D C, Wang,J, Moldoveanu, Z, McPherson, S and Morrow, C, 1997. Immunization of micewith poliovirus replicons expressing the C-fragment of tetanus toxinprotects against lethal challenge with tetanus toxin. Vaccine15:257-264).

Both humoral antibody and cellular immune responses are implicated inprotection and recovery from flavivirus infection. The flavivirusreplicon-based vaccine of interest induces both arms of the immuneresponse. The particles are composed of preM and E proteins and thus,the particles themselves are immunogens. But the particles of interestinfect host cells, and in those cells, additional preM and E proteinsare expressed. The preM and E proteins either can be released from thosecells, providing additional antigenic stimuli to the host, or can beexpressed at the infected host cell surface, for example, on hostantigen presenting cells, to provide yet another antigenic stimulus tothe host.

The vaccine replicons contain the viral antigens, including structuralproteins prM and E, and optionally can express nonstructural proteinNS1. Previous reports demonstrated that those viral proteins induce aprotective immune response (Heinz, F X & Roehrig, J, 1990, inImmunochemistry of Viruses, Vol II. Amsterdam-NY-Oxford, Elsvier, p.289-305; Heinz F X, 1986, Epitope mapping of flavivirus glycoproteins.Adv Virus Res 31:103-168; Bray, M and Lai, C J, 1991, Dengue viruspremembrane and membrane proteins elicit a protective immune response.Virology, 185:505-508; Henchal E A, Henchal, L S and Shlesinger, J J,1988, Synergistic interactions of anti-NS1 monoclonal antibodies protectpassively immunized mice from lethal challenge with dengue 2 virus. J.Gen Virol. 69: 2101-2107; and Schlesinger J J, Brandriss, M W, Cropp, CB et al., 1986. Protection against yellow fever in monkeys byimmunization with yellow fever virus non-structural protein NS1. J Virol60:1153-1155).

Suitable models for testing the efficacy of a pharmaceutical compositionof interest exist as there are animal models that simulate flavivirusinfection. For example, a protocol used for immunization of mice andsubsequent challenge with dengue virus has been described (Bray, M,Zhao, B, Marckoff, L, Eckels, K, Chanock, R M and Lai, C, 1989, Miceimmunized with recombinant vaccinia virus expressing dengue 4 virusstructural proteins with or without nonstructural protein NS1 areprotected against fatal dengue encephalitis. J. Virol. 63:2853-2856).Briefly, in 10 mice for each group, female BALB/c mice are immunized at3 weeks of age (day 1) and again on day 14 by intraperitonealinoculation of the virus-like particles. Control animals receivephosphate-buffered saline (PBS). All animal are bled on day 0 and day21. Mice are challenged on day 22 by intracerebral injection of 100times the 50% lethal dose (LD₅₀) of dengue virus. Following challenge,mice are observed for 21 days for signs of encephalitis, and the numberof mice with any important symptoms (encephalitis, paralysis and death)are recorded daily. Sera also are collected from survivors forcomparison with prechallenged sera.

The dose of each VLP is determined. The seroresponse of immunized miceto individual dengue virus proteins is analyzed byradioimmunoprecipitation of [³⁵S] methionine-labeled dengue virusantigens using commercially available labeling kits and antibodies. Theplaque reduction assay is used to measure titers of dengue-specificneutralizing antibodies in mouse sera. Approximately 0.5 ml of a serumsample to be used in the assay first is heat inactivated by incubationfor 30 min at 56° C. Fourfold dilutions of serum in a final volume of0.3 ml, starting at 1:10 dilution, are prepared using medium M199 with2% heat-inactivated FBS as a diluent. To each 0.3 ml aliquot of dilutedserum is added an equal volume of medium containing 150-180 PFU ofdengue virus. Virus and serum are mixed and incubated for 30 min at 37°C. In each assay, a no serum control and controls consisting of eachdengue type-specific mouse hyperimmune ascitic fluid (ATCC) at twodilutions are also included. Virus-serum and control mixtures are platedon confluent monolayers of LLC-MK2 cells in Costar six-well plates(Corning Inc., Corning, N.Y.) at 0.2 ml/well. Duplicate wells areinfected for each sample. Virus adsorption is carried out for 1 hour atroom temperature with manual rocking every 15 min. Wells then areoverlaid with medium containing 1% agarose (SeaKem L E; BioWhittaker,Rockland, Me.) in Earle's balanced salt solution plus 10% FBS with addedessential vitamins and amino acids (Invitrogen) at 6 ml/well. Plates areincubated for 7 days at 37° C. in 5% CO₂. Wells are then overlaid with4% neutral red solution (4 ml of neutral red solution plus 96 ml of PBS)containing 1% agarose. Plates are incubated for 24 hours at 37° C. Theaverage of plaque counts is used to calculate the 50% reduction inplaque number level.

An exemplified embodiment is a dengue vaccine that is tetravalent, onethat immunizes a host to dengue types 1, 2, 3 and 4.

Because the flaviviruses have a unified generic genetic structure, thegoal of having a tetravalent vaccine is achieved by the use of repliconsas taught herein for each of the types 1, 2, 3 and 4. Moreover, themethod of making a vaccine as taught herein using two vectors carryingthe flavivirus structural genes and a packaging cell carrying a geneencoding an overlapping peptide that in yeast following homologousrecombination reconstitutes the whole of the flavivirus structuralgenes, has application not only to dengue virus but also to allflavivirus species as well.

The PVLP of interest is incorporated into pharmaceutical compositionssuitable for administration to serve as a vaccine, as known in thevaccine art. Such compositions typically comprise the active ingredientand a pharmaceutically acceptable carrier. As used herein, the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents and the like, compatiblewith pharmaceutical administration. The use of such media and agents forpharmaceutically active substances is well known in the art. Exceptinsofar as any conventional media or agent is incompatible with theactive compound, use thereof in the compositions is contemplated.Supplementary active compounds also can be incorporated into thecompositions.

A pharmaceutical composition of the invention for use as disclosedherein is formulated to be compatible with the intended route ofadministration. Examples of routes of administration include parenteral,e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation),transdermal (topical), transmucosal and rectal administration. Solutionsor suspensions used for parenteral, intradermal or subcutaneousapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerin, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as EDTA; buffers such as acetates, citrates or phosphates andagents for the adjustment of tonicity such as sodium chloride ordextrose. pH can be adjusted with acids or bases, such as HCl or NaOH.The parenteral preparation can be enclosed in ampoules, disposablesyringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL® (BASF; Parsippany, N.J.) or phosphate-buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. The composition must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol andliquid polyethylene glycol and the like) and suitable mixtures thereof.The proper fluidity can be maintained, for example, by the use of acoating such as lecithin, by the maintenance of the required particlesize in the case of dispersion and by the use of surfactants. Preventionof the action of microorganisms can be achieved by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,ascorbic acid, thimerosal and the like. In many cases, it will bepreferable to include isotonic agents, for example, sugars, polyalcoholssuch as mannitol, sorbitol or sodium chloride in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent that delays absorption, forexample, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze drying that yield a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. The composition can be enclosed in gelatin capsules orcompressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches or capsules. Oral compositionsalso can be prepared using a fluid carrier to yield a syrup or liquidformulation, or for use as a mouthwash, wherein the compound in thefluid carrier is applied orally and swished and expectorated orswallowed.

Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate or orange flavoring.

For administration by inhalation, the compound is delivered in the formof, for example, an aerosol spray from a pressurized container ordispenser that contains a suitable propellant, e.g., a gas such ascarbon dioxide or a nebulizer, or a mist.

Systemic administration also can be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants generally are known in the art and include, for example,for transmucosal administration, detergents, bile salts and fusidic acidderivatives. Transmucosal administration can be accomplished through theuse of nasal sprays or suppositories. For transdermal administration,the active compounds are formulated into ointments, salves, gels orcreams as generally known in the art.

The vaccine also can be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compound is prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters and polylactic acid.

Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials also can be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Liposomal suspensions (including liposomes targeted with monoclonalantibodies and other such targeting molecules) also can be used aspharmaceutically acceptable carriers. Those can be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The dosages, forexample, preferred route of administration and amounts, are obtainablebased on empirical data obtained from preclinical and clinical studies,practicing methods known in the art. For repeated administrations overseveral days or longer, depending on the condition, the treatment issustained until a desired suppression of disease symptoms occurs.However, other dosage regimens may be useful. The progress of thetherapy is monitored easily by conventional techniques and assays. Anexemplary dosing regimen is disclosed in WO 94/04188. The specificationfor the dosage unit forms of the invention is dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved and the limitationsinherent in the art of compounding such an active compound for thetreatment of individuals.

The pharmaceutical compositions can be included in a container, pack ordispenser together with instructions for administration.

Another method of administration comprises the addition of a compound ofinterest into or with a food or drink, as a food supplement or additive,or as a dosage form taken on a prophylactic basis, similar to a vitamin.The peptide of interest can be encapsulated into forms that will survivepassage through the gastric environment. Such forms are commonly knownas enteric-coated formulations. Alternatively, the peptide of interestcan be modified to enhance half-life, such as chemical modification ofthe peptide bonds, to ensure stability for oral administration, as knownin the art.

All references cited herein are herein incorporated by reference inentirety.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present invention andwithout diminishing its intended advantages. It is therefore intendedthat such changes and modifications be covered by the appended claims.

The invention is claimed as follows:
 1. A non-replicating flavivirusparticle comprising a recombinant replicon that expresses at least oneimmunologic determinant of a flavivirus envelope protein.
 2. Theparticle of claim 1, wherein said flavivirus particle comprises a denguevirus.
 3. The particle of claim 1, wherein said flavivirus particlecomprises a West Nile virus.
 4. The particle of claim 1, wherein saidflavivirus particle comprises a Japanese encephalitis virus.
 5. Theparticle of claim 1, wherein said flavivirus particle comprises a yellowfever virus.
 6. The particle of claim 1, wherein said replicon does notexpress a capsid protein.
 7. The particle of claim 1, wherein saidreplicon expresses at least one immunologic determinant of a premembraneor a membrane protein of said envelope protein.
 8. A pharmaceuticalcomposition comprising the particle of claim 1 and a pharmaceuticallyacceptable carrier, diluent or excipient.
 9. A method of making theparticle of claim 1, comprising transforming a host cell with saidreplicon, then transforming said host cell with a replicon thatexpresses genes that complement said replicon of claim 1 and isolatingparticles produced by said host cell.
 10. The method of claim 9, whereinsaid complementing replicon comprises Sindbis virus C, prM and/or E openreading frames.
 11. A method of making the particle of claim 1,comprising transforming a host cell with said replicon, wherein saidreplicon does not express a gene product essential for making aninfectious virus particle, wherein said host cell expresses said geneproduct essential for making an infectious gene particle, and isolatingparticles produced by said host cell.
 12. The method of claim 11,wherein said host cell expresses a C protein.
 13. The method of claim12, wherein expression of said C protein is induced when said host cellis transformed with said replicon.
 14. A transformed cell that expressessaid flavivirus envelope protein of claim
 1. 15. The transformed cell ofclaim 14, further comprising a flavivirus replicon that does not expressa C protein or expresses a C protein that cannot comprise a capsid of aninfectious virus particle.